Learning from the enemy

Viruses are the ultimate hackers of biological systems. Synthetic biologists might begin to catch up in a billion years or so, depending, of course, on how strong the evolutionary pressures on them are. But for now, for frighteningly elegant and complex interventions in cellular behavior, viruses are hard to beat. And that means that when you find a virus messing with your system, you can learn a lot from how it achieves its effects.

A recent paper (Maynard et al. 2012. Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting. Mol. Sys. Biol. 8; 567) dissects a case in point. In earlier work, this group identified some pathways in E. coli that rather unexpectedly affected the efficiency of lambda phage replication. For example, knocking out members of the 2-thiouridine synthesis pathway inhibited replication; conversely, knocking out members of a pathway involved in making iron-sulfur clusters increased replication. These are both pathways that use sulfur, and so it was natural to suspect that the two activities are related, though both mechanisms were unknown.

What is 2-thiouridine good for? One of its uses is to modify certain tRNAs (those that accept Lys, Glu and Gln as payloads) with a thiol group, providing a clue that something to do with translation might be involved. In fact, thiolation of these tRNAs is important for reducing ribosomal frame-shifting. You might think that this is an unlikely place to look for effects on the virus. But you’d be wrong. It turns out that many viruses, including HIV, use a lovely strategy called programmed ribosomal frameshifting to make themselves more efficient by producing two proteins from one gene. It works like this: when the ribosome reaches a so-called “slippery sequence”, it — um — slips, either backwards or forwards. When the ribosome slips, it then misses a stop codon further along in the gene, so one protein is made that stops at the stop codon and the other is made as a result of read-through. The ratio between the proteins is determined by the frequency of slippage, and the ratio matters because the two proteins have different functions. In the case of lambda, the proteins made in this slippery fashion are called gpG and gpGT, and they seem to act as chaperones for the asssembly of the phage’s tail.

Maynard et al. show that knocking out the tRNA thiolating enzymes increases frameshifting, and also decreases viral replication. In lambda, the slippery sequence reads G-GGT-TTG: without frameshifting, you read GGT (Gly) and TTG (Lys), and later hit a stop codon, resulting in the shorter protein, gpG. With a -1 frameshift, you read GGG (Gly) and TTT (Lys) — the same amino acid sequence, but now you miss the stop codon and read through to get gpGT. Both the TTG and the TTT codons are read by the same Lys tRNA, with the anticodon UUU, which is one of the tRNAs that is normally thiolated. It seems that removing the thiolation leads to too much frameshifting and thus a non-ideal ratio of gpGT to gpG.

So far so good: but why does knocking out the iron-sulfur cluster pathway also affect replication? Iron-sulfur clusters do a lot of things, but with the example of the thiolation pathway to guide them the authors focused their attention on translation. It turns out that iron-sulfur cluster containing enzymes are also important for modifying tRNAs, this time using 2-thiocytidine. Both types of modifications are important for reducing frameshifting, but apart from that the only connection between them is that they get their sulfur from the same place, a cysteine desulfurase called IscS. Given all this, you might expect that knocking down this 2-thiocytidine pathway would also lead to increased frameshifting and increased levels of gpGT. If this turned out to be true, we would be left with the problem of why increasing frameshifting via knockout of the thiolation pathway has the opposite effect on viral replication from increasing frameshifting via the iron-sulfur pathway, but we could probably handwave our way out of that by thinking about effects of different size. We shovel similar contradictions under the rug every day, in biology.

But in fact the effect is in the other direction. The authors show that knocking out a key component of the 2-thiocytidine pathway dramatically decreases the level of gpGT. While this solves the problem of the opposing effects, it leaves us with a new puzzle: removing a modification that improves ribosomal fidelity actually reduces frameshifting. At this point the authors abandoned Occam’s razor, and started to consider the possibility of interactions between the two apparently independent pathways they were studying. Here’s a schematic of the two pathways. The red protein is IscU, in the iron-cluster pathway; it’s colored red because knocking out the relevant gene leads to increased viral replication, in other words, the protein has a negative effect on replication. The green protein is TusA, in the thiolation pathway. Knocking out the tusA gene decreases viral replication, so the protein has a positive effect on replication.

From Maynard et al.

Here’s the hypothesis the authors set out to examine: tRNA thiolation (which depends on TusA; green blobs) keeps frameshifting at a level that is OK for lambda replication, but somewhat higher than ideal. Knocking out TusA increases frameshifting and therefore causes reduced virus replication. IcsU and TusA compete for sulfur delivered by IscS. When you knock out IscU (red blobs), you relieve this competition, so you get more sulfur flowing down the TusA pathway. This reduces frameshifting to a level that is closer to ideal, and the virus does even better.

To gain insight into whether this hypothesis could explain their experimental results, Maynard et al. developed a model of the pathway, and combined it with an earlier model they had made of virus replication in E. coli. The replication model predicts, for example, the rate of replication of the virus in cells, the likelihood that the virus will trigger lysis, and the number of functional progeny released. With the combined model, they could predict the time course and extent of virus replication in cells with various pathway modifications. To test their predictions they introduced a couple of additional manipulations: in one case they put IcsU production was controlled by an IPTG-inducible promoter, in another they made double mutants lacking both TusA and IcsU. For comparison they also built a second model that assumed that the TusA and IcsU effects on virus were independent.

Let us remember, for otherwise Johan Paulsson will undoubtedly remind us, that just because mathematical models are very detailed doesn’t mean that a match between theory and experiment “proves” the model. Your experiments might simply be inadequate to distinguish between a mistaken model and reality. That said, the results of Maynard et al.’s experiments were clearly not consistent with the “independent effects” model — this model fails to predict the behavior of the double mutant — while the simulated results of the “competition for sulfur model” were pretty close to the experimental reality.

The picture that emerges from all this is one of surprising fragility in basic elements of the translational machinery — what other forms of competition for sulfur might induce increased frameshifting? And could we use the fact that the behavior of tRNAs is unexpectedly manipulatable as an antiviral strategy? If we were only talking about lambda phage, that might not be a very exciting question, but viruses such as HIV and the virus that causes SARS use ribosomal frameshifting as well. So, just possibly, this new angle on how viruses manipulate cellular machinery to suit themselves may lead to a new potential strategy to frustrate them.

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